Modulation of the mitochondrial permeability transition pore complex in GSK-3β-mediated myocardial protection

Abstract

Recently we found that the level of anti-infarct tolerance afforded by ischemic preconditioning (IPC) and erythropoietin (EPO) infusion was closely correlated with the level of Ser9-phospho-GSK-3β upon reperfusion in the heart. To get an insight into the mechanism by which phospho-GSK-3β protects the myocardium from ischemia/reperfusion injury, we examined the effects of IPC and EPO on interactions between GSK-3β and subunits of the mitochondrial permeability transition pore (mPTP) in this study. Rat hearts were subjected to 25-min global ischemia and 5-min reperfusion in vitro with or without IPC plus EPO infusion (5 units/ml) before ischemia. Ventricular tissues were sampled before or after ischemia/reperfusion to separate subcellular fractions for immunoblotting and immunoprecipitation. Reperfusion increased mitochondrial GSK-3β by 2-fold and increased phospho-GSK-3β level in all fractions examined. Major subunits of mPTP, adenine nucleotide translocase (ANT) and voltage-dependent anion channel (VDAC), were co-immunoprecipitated with GSK-3β after reperfusion. Phospho-GSK-3β was co-immunoprecipitated with ANT but not with VDAC. IPC+EPO significantly increased the levels of GSK-3β and phospho-GSK-3β that were co-immunoprecipitated with ANT to 145±8% and 143±16%, respectively, of baseline but did not induce phospho-GSK-3β–VDAC binding. A PKC inhibitor and a PI3 kinase inhibitor suppressed the IPC+EPO-induced increase in the level of phospho-GSK-3β–ANT complex. The level of cyclophilin D co-immunoprecipitated with ANT after reperfusion was significantly reduced to 39±10% of the control by IPC+EPO. These results suggest that reduction in affinity of ANT to cyclophilin D by increased phospho-GSK-3β binding to ANT may be responsible for suppression of mPTP opening and myocardial protection afforded by IPC+EPO.